Ecotoxicological Properties of Titanium Dioxide Nanomorphologies in Daphnia magna

In this work, the structural, vibrational, morphological, and colloidal properties of commercial 15.1 nm TiO2 nanoparticles (NPs) and nanowires (NWs, 5.6 thickness, 74.6 nm length) were studied with the purpose of determining their ecotoxicological properties. This was achieved by evaluating acute ecotoxicity experiments carried out in the environmental bioindicator Daphnia magna, where their 24-h lethal concentration (LC50) and morphological changes were evaluated using a TiO2 suspension (pH = 7) with point of zero charge at 6.5 for TiO2 NPs (hydrodynamic diameter of 130 nm) and 5.3 for TiO2 NWs (hydrodynamic diameter of 118 nm). Their LC50 values were 157 and 166 mg L−1 for TiO2 NWs and TiO2 NPs, respectively. The reproduction rate of D. magna after fifteen days of exposure to TiO2 nanomorphologies was delayed (0 pups for TiO2 NWs and 45 neonates for TiO2 NPs) in comparison with the negative control (104 pups). From the morphological experiments, we may conclude that the harmful effects of TiO2 NWs are more severe than those of 100% anatase TiO2 NPs, likely associated with brookite (36.5 wt. %) and protonic trititanate (63.5 wt. %) presented in TiO2 NWs according to Rietveld quantitative phase analysis. Specifically, significant change in the heart morphological parameter was observed. In addition, the structural and morphological properties of TiO2 nanomorphologies were investigated using X-ray diffraction and electron microscopy techniques to confirm the physicochemical properties after the ecotoxicological experiments. The results reveal that no alteration in the chemical structure, size (16.5 nm for TiO2 NPs and 6.6 thickness and 79.2 nm length for NWs), and composition occurred. Hence, both TiO2 samples can be stored and reused for future environmental purposes, e.g., water nanoremediation.


Introduction
Promising metal/metal-oxide nanomaterials have been synthesized by different chemical, physical, and biological synthesis techniques [1,2], such as pulse laser ablation, highenergy ball milling, sputtering, chemical reduction, microemulsion, sol gel, and green synthesis, to create different shapes of nanoparticles (NPs) [1,2]. Therefore, different enhanced physicochemical properties are expected and will differ in the required applications/purposes [2]. One of these applications is to solve existing environmental problems such as the accelerated consumption of fossil fuels through the removal of emerging pollutants that will end up in aquatic ecosystems, etc. [1,3,4]. One important nanomaterial is titanium dioxide (TiO 2 ), the world production of which is estimated to reach 2.5 million tons in 2025 [5]. These are found in applications such as sun creams and self-cleaning products due to their photocatalytic, antimicrobial, and UV protection properties [6,7]. Hence, due to the high production of these nanomaterials at industrial levels, it is expected that they will be spread to aqueous bodies and represent a toxic material to water specimens as well. The ultimate process for these nanomaterials in aquatic ecosystems is a current concern and previous toxic effect evaluations have been reported [8][9][10]. experiments regarding the structural and morphological characterization of the recovered nanosystems were undertaken by X-ray diffraction (XRD), selected area electron diffraction (SAED), and electron energy loss spectroscopy (EELS) to ensure their reusability and regeneration properties.

Chemicals
To perform the ecotoxicological experiments in the presence of the environmental bioindicator D. magna, both 20 nm anatase TiO 2 NPs and TiO 2 NWs (10 µm in length and 10 nm in thickness) were purchased from SIGMA-ALDRICH without any other chemical modifications. Both samples were synthesized by the hydrothermal method.

Characterization of the TiO 2 Samples
X-ray diffraction data were obtained using an Empyrean diffractometer operating with CuK α , 45 kV and 40 mA. X-ray diffractograms were collected in step scanning mode, 2θ = 10-100 • , step of 0.026 • , and 20 s/step. By means of the Match v3 software [19], the phase identification and indexation of both samples was carried out, resulting in a monophasic sample (anatase with a crystallographic chart #500-0024) for the NPs and a biphasic sample (brookite with a crystallographic chart #900-9088 and protonic trititanate with a crystallographic chart crystallography #433-6946) for the NWs. The Rietveld refinement was applied from the crystallographic information file (cif) of both samples using the FullProf Suite software (Gif sur Yvette Cedex, France, version January 2021). To refine the X-ray diffraction profile, the Thompson-Cox-Hastings (TCH) pseudo-Voigt axial divergence asymmetry function was used. The instrumental resolution function (IRF) of the diffractometer was obtained from an aluminum-oxide (Al 2 O 3 ) standard with refined Caglioti parameters, U = 0.0093, V = −0.0051, and W = 0.0013 [20,21].
The µ-Raman spectrum was measured in a Renishaw inVia Raman microscope. A wavelength of λ = 785 nm (initial laser power of 82.8 mW) was used as the excitation source. An optical objective of 50× magnification was chosen for the measurements. The Raman mode profiles (spectrum taken at laser power fraction of 10% over the sample) were fit using Lorentzian functions.
The colloidal properties by means of effective hydrodynamic diameter and Zeta potential of the TiO 2 suspensions (both morphologies) were determined using a Brookhaven Nanobrook 90 Plus PALS. The zeta potential of the suspensions as a function of pH was obtained for TiO 2 samples to find the point of zero charge. In addition, the hydrodynamic diameter at different pH values (2,5,7,9,12) were measured for initial 50 mg L −1 TiO 2 NPs and NWs. All the experiments were done in duplicate. The base line index criterium was used to analyze the correct measurement.
To determine the average particle size, particle size distribution, and morphological features, the 200 kV JEOL 2100F (Tokyo, Japan) imaging electron microscopy equipment was employed in transmission (TEM), scanning (STEM), and high-resolution modes. For particle size distribution (PSD) estimation, a total number of particles and wires of 800 to 1000 was counted from 30-35 images using the Image J software. A log-normal distribution was considered to fit the obtained histograms according to [22]. Finally, the polydispersity values were estimated from the standard deviation of the log-normal distribution. The microscope has two accessories for energy-dispersive X-ray spectroscopy (EDS) and electron energy loss spectroscopy (EELS) (EELS-GIF Tridiem GATAN). Both techniques were used to evaluate the atomic composition at the nanoscale using the mode STEM. EELS experiments were carried out in the STEM imaging mode using a spot size of 0.7 nm. The spectrometer aperture was 5 mm and the energy resolution (measured by the FWHM of the zero-loss peaks) was approximately 1.8 eV. For the morphological analysis, commercial samples without any modification were labeled as before samples and recovered samples after ecotoxicological experiments were labeled as after. This nomenclature was also used ahead in this work.

D. magna Culture
The set of D. magna individuals used for the ecotoxicity experiments were cultivated in the laboratory under optimized conditions which resemble their habitat. D. magna were maintained in an 8:16 h light:dark photoperiod and a temperature of (20 ± 1) • C and pH = 7.5 ± 0.5. The required hatchlings were obtained by separating 120 adult daphnia individuals with the potential to give hatchlings in a short time. The daphnia individuals were fed daily with microalgae of the genus Scenedesmus, in relation to the volume of the beaker, with a scale of 1 mL per 100 mL [12].
These D. magna individuals were separated in to four beakers with 200 mL of standardized water for the cultivation of D. magna, after one to five days the corresponding neonates were born, which are clones due to the favorable conditions in which they were found. Neonates are D. magna individuals with a life span of less than 24 h. If daphnia are well fed, they can have up to 65 hatchlings in a single litter, with an average size of 12 hatchlings per litter [23].
When the 120 D. magna individuals were separated in the four reproduction beakers no more hatchlings were generated. Then, they were returned to the original culture beaker and other D. magna with the potential to give hatchlings in a short time were separated again with the purpose of continuing the ecotoxicity experiment.
It should also be highlighted that the set of D. magna must have a specific culture temperature, since being a bioindicator, it is very sensitive to sudden physicochemical changes that occur in its habitat. For example, a sudden change in temperature inside the beaker during feeding, oxygen concentration, etc. [23], under the specific laboratory conditions.

D. magna Exposure Protocol
The TiO 2 concentrations for the ecotoxicity experiment were done separately. In a volume of 200 mL of standardized water, five concentrations of TiO 2 NPs were prepared: 37.5 mg L −1 , 75 mg L −1 , 150 mg L −1 , 300 mg L −1 , and 600 mg L −1 . While for the TiO 2 NWs five chosen concentrations were also prepared: 50 mg L −1 , 100 mg L −1 , 200 mg L −1 , 400 mg L −1 , and 800 mg L −1 . The individuals for each concentration were 10 neonates of D. magna. Experiments were done in duplicate and the mean values were reported.

Morphological Analysis of D. magna
The morphological evaluation was undertaken after 16 days, one day for the exposure with the TiO 2 samples. After the 24-h exposure, surviving D. magna were moved to another beaker with conditions prior to TiO 2 exposure. The remaining 15 days were for the control of the surviving D. magna. On the last day of the experiment, the length (micrometers) of the tail, body, antenna, eye, and heart morphological parameters of the surviving D. magna were measured using an optical microscope and ToupView software on a computer.
Within two weeks, D. magna reaches sexual maturity (generally between the sixth and ninth day [23]) to be able to reproduce either sexually or asexually depending on habitat conditions. In the ecotoxicity experiment it was evidenced that the negative control D. magna began to have newborns from the ninth day.
Finally, the statistical analysis of the morphological parameters was analyzed by means of the paired Student's t-test using the p value criterium of 0.05 for the corresponding significance. A box plot was used to visualize the comparison of morphological parameters between exposed daphnia and negative control D. magna. These calculations were made using SPSS 27 statistical software. The comparison was made with respect to the negative control, a set of D. magna that were not exposed to the TiO 2 sample.

Rietveld Refinement Analysis
Refined X-ray diffractograms are presented in Figure 1 for TiO 2 NPs (A) and TiO 2 NWs (B). The refined X-ray diffractogram of commercial TiO 2 NPs, Figure 1A, consists of the reported polymorph phase of TiO 2 , anatase, synthesized by sol-gel method assisted with Nanomaterials 2023, 13, 927 5 of 22 heat treatment [24]. The corresponding Bragg's angle positions for crystalline identified phases are given in Table S1.

µ-Raman Analysis
µ -Raman spectrum for commercial TiO2 NPs has been previously characteriz [20]. To identify the vibrational Raman bands for the TiO2 NWs sample in Figure 2, linear regression was performed by means of a finite sum of Lorentzian function reason the Lorentzian was taken as the profile function is because it better approxi the physical process in which inelastic light scattering intervenes [27].
Reviewing the corresponding literature, some Raman peaks or vibrational ban the Raman curve of the TiO2 NWs sample were identified and are summarized in T [28][29][30]. In the X-ray diffractogram of Figure 1B, two crystalline contributions were observed for TiO 2 NWs, the crystalline phase percentage of the main crystalline phase (Protonic Trititanate, 63.5%) is higher than the secondary identified crystalline phase (Brookite, 36.5%). On the other hand, as was explained in [25], the second crystalline phase is a product of the ion exchange that occurs in sodium trititanate, wherein the sodium is eliminated at a temperature of 120 • C, leaving the protonic trititanate available. Their respective Miller's indices and Bragg's angles were found at: (200) at 11.6 • , (110) at 24 • , and (020) at 48 • [25]. These Bragg's angles, according to [25], appear when the calcination temperature is lower than 350 • C. However, when the temperature is higher than 550 • C, the Bragg's angle at 11.6 • completely disappears. The disappearance of Bragg's peak (200) is attributed to dehydration caused by the increase in temperature [26]. In this case, a Bragg's peak with Miller index (211) appears at 43.9 • . Despite the fact that the synthesis method in [26] was also hydrothermal, the two employed precursors of titanium dioxide (rutile and anatase) together with a solution of sodium hydroxide (NaOH) [26] were different and we can discard the idea of high temperature synthesis due to the lack of this (211) Miller index. Hence, the temperature for hydrothermal synthesis of the TiO 2 NWs was less than 350 • C. Moreover, the detail indexation of brookite phase is give in Table S2.

µ-Raman Analysis
µ-Raman spectrum for commercial TiO 2 NPs has been previously characterized in [20]. To identify the vibrational Raman bands for the TiO 2 NWs sample in Figure 2, a nonlinear regression was performed by means of a finite sum of Lorentzian functions. The reason the Lorentzian was taken as the profile function is because it better approximates the physical process in which inelastic light scattering intervenes [27].  The pure brookite phase presents between four and five characteristic vibrational Raman bands [31,32]. The strong optical signal for brookite is positioned at ~150 cm −1 and no other peaks were found. This is because brookite is a secondary phase and major chemical compositions are detected for the H2Ti3O7, which agrees with Rietveld quantitative analysis. It is worth mentioning that that the phonon mode located at 280 cm −1 is a characteristic vibrational band of trititanate nanotube geometries [33].

DLS Technique and Point of Zero Charge (p.z.c.) Determination
The effective hydrodynamic diameter (EHD) of the TiO2 NPs sample was determined using the DLS technique, in which the pH value of the TiO2 sample was varied to obtain different values of this EHD. In Figure S1, one can see the EHD for five corresponding pH values. Since TiO2 NWs do not have a spherical geometry, the Stokes-Einstein equation cannot, in principle, be applied, since it is valid for spherical particles only [34]. However, since they are in a fluid, they tend to agglomerate with other nanowires until they form a quasi-spherically symmetrical particle that agree with the DLS principle. The EHD measured at a pH equal to 7 of the TiO2 NWs was 118 nm with a base line index of 10. Reviewing the corresponding literature, some Raman peaks or vibrational bands of the Raman curve of the TiO 2 NWs sample were identified and are summarized in Table 1 [28][29][30]. The pure brookite phase presents between four and five characteristic vibrational Raman bands [31,32]. The strong optical signal for brookite is positioned at~150 cm −1 and no other peaks were found. This is because brookite is a secondary phase and major chemical compositions are detected for the H 2 Ti 3 O 7 , which agrees with Rietveld quantitative analysis. It is worth mentioning that that the phonon mode located at 280 cm −1 is a characteristic vibrational band of trititanate nanotube geometries [33].

DLS Technique and Point of Zero Charge (p.z.c.) Determination
The effective hydrodynamic diameter (EHD) of the TiO 2 NPs sample was determined using the DLS technique, in which the pH value of the TiO 2 sample was varied to obtain  Figure S1, one can see the EHD for five corresponding pH values. Since TiO 2 NWs do not have a spherical geometry, the Stokes-Einstein equation cannot, in principle, be applied, since it is valid for spherical particles only [34]. However, since they are in a fluid, they tend to agglomerate with other nanowires until they form a quasi-spherically symmetrical particle that agree with the DLS principle. The EHD measured at a pH equal to 7 of the TiO 2 NWs was 118 nm with a base line index of 10.
The p.z.c. is the pH value corresponding to a null zeta potential value [35], or also understood as an electrical surface potential equal to zero (shear plane model). At lower pH values (below p.z.c.) it is positively charged, while at higher pH (above p.z.c.) it is negatively charged [35]. As reported by Calle et al. the p.z.c. of TiO 2 (anatase 70% and rutile 30%) was found to be 6.5, whilst doped TiO 2 samples have a low p.z.c. [36]. This value of 6.5 is equal to that found with our titration experiment, see Figure 3A,B, also 6.5, for anatase TiO 2 NPs. However, for compounds that have impurities, the p.z.c. is less than 6.5. This may be the reason that the mean p.z.c. for the TiO 2 NWs sample is 5.3, see Figure 3C,D, since it is a biphasic sample (brookite and protonic trititanate).
Nanomaterials 2023, 13, x FOR PEER REVIEW 8 o value of 6.5 is equal to that found with our titration experiment, see Figure 3A,B, also for anatase TiO2 NPs. However, for compounds that have impurities, the p.z.c. is less t 6.5. This may be the reason that the mean p.z.c. for the TiO2 NWs sample is 5.3, see Fig  3C,D, since it is a biphasic sample (brookite and protonic trititanate).

TEM Analysis
Figures 4a,b,d,e and 5a,b show the TEM images, while Figures 4c,f and 5c-f t respective particle size distribution (PSD) histograms for TiO2 NPs and TiO2 NWs, obtained statistical parameters after fitting with a normal and log-normal distribution listed in Table 2. This was achieved with the aim of comparing with the manufactur measurements (Sigma-Aldrich) that reported 20 nm for mean particle size, in case of T NPs, and 10 nm (thickness)/10 µ m(length) for TiO2 NWs. For the case of TiO2 NPs, difference between before (15.1 nm) and after (16.5 nm) did not show a noticeable dif ence with respect to the agglomeration of TiO2 NPs, this was also notice in the interpla distance of 3.5 Å for (101) and 2.4 Å for (004) planes, in agreement with Rietveld ref ment, cif files in Section 2.2, and supporting information in Tables S1 and S3. Howe some morphological changes to the measurements reported by the manufacturer w found in the thickness and length parameters (before exposure) for TiO2 NWs. The   Table 2. This was achieved with the aim of comparing with the manufacturer's measurements (Sigma-Aldrich) that reported 20 nm for mean particle size, in case of TiO 2 NPs, and 10 nm (thickness)/10 µm(length) for TiO 2 NWs. For the case of TiO 2 NPs, the difference between before (15.1 nm) and after (16.5 nm) did not show a noticeable difference with respect to the agglomeration of TiO 2 NPs, this was also notice in the interplanar distance of 3.5 Å for (101) and 2.4 Å for (004) planes, in agreement with Rietveld refinement, cif files in Section 2.2, and supporting information in Tables S1 and S3. However, some morphological changes to the measurements reported by the manufacturer were found in the thickness and length parameters (before exposure) for TiO 2 NWs. The estimated thickness was less than 10 nm and the mean length value was 74.6 nm. These values remain close after performing the ecotoxicological experiments. The small value for the reported length can be related to the small temperature used for the synthesis (<350 • C), as discussed in Section 3.1. It is worth mentioning the importance of performing a rigorous characterization to correctly determine the LC 50 and morphological D. magna changes, as we will discuss in the following sections. Table 2. TEM parameters obtained for the TiO 2 samples after fitting the data with normal and log-normal distribution. <D> is the mean particle size.

Samples <D> (nm) Standard Deviation Polydispersity
Before characterization to correctly determine the LC50 and morphological D. magna changes, as we will discuss in the following sections.

Acute Toxicity of TiO2 in D. magna
Over the course of 24 h, D. magna neonates were exposed to five different concentrations of TiO2 NPs and NWs. 0% of mortality was found for 75 mg L −1 (TiO2 NPs) and 800

Acute Toxicity of TiO 2 in D. magna
Over the course of 24 h, D. magna neonates were exposed to five different concentrations of TiO 2 NPs and NWs. 0% of mortality was found for 75 mg L −1 (TiO 2 NPs) and 800 mg L −1 (TiO 2 NWs). The surviving daphnias were assessed for an additional 15 days in a beaker with conditions before exposure in a volume of 200 mL of daphnia water to determine what toxic effects they may have encountered (morphological and reproduction rate experiments).

Lethal Concentration 24-h LC 50
As previously mentioned, 10 neonates of D. magna for concentration were used for 24-h LC 50 determination. Modified Probit analysis was used to calculate the 24-h LC 50 for TiO 2 samples. Figure 6A,C show mortality (%) as a function of concentration (mg L −1 ) for both TiO 2 samples.

Lethal Concentration 24-h LC50
As previously mentioned, 10 neonates of D. magna for concentration were used for 24-h LC50 determination. Modified Probit analysis was used to calculate the 24-h LC50 for TiO2 samples. Figure 6A,C show mortality (%) as a function of concentration (mg L −1 ) for both TiO2 samples.  In Figure 6B, a cubic regression was performed on the experimental data. The nonlinear regression has a goodness of fit of 1.0 and has the following equation: In this case, substituting the value of 5.0, which is the probit value of 50% mortality, into Equation (1) gave us two points of intersection, two values (positive and negative) for the LC50. These points lie at the probit numerical values of 1.58 and 2.22. Therefore, the positive value that lies in the LC50 range of various previous investigations [8][9][10]37,38] for TiO2 NPs was chosen. Hence, the concentration at which the LC50 occurs was 166 mg L −1 .
In Figure 6D, a quadratic regression was performed on the experimental data. The non-linear regression has an R 2 = of 0.859 and has the following equation: In Figure 6B, a cubic regression was performed on the experimental data. The nonlinear regression has a goodness of fit of 1.0 and has the following equation: In this case, substituting the value of 5.0, which is the probit value of 50% mortality, into Equation (1) gave us two points of intersection, two values (positive and negative) for the LC 50 . These points lie at the probit numerical values of 1.58 and 2.22. Therefore, the positive value that lies in the LC 50 range of various previous investigations [8][9][10]37,38] for TiO 2 NPs was chosen. Hence, the concentration at which the LC 50 occurs was 166 mg L −1 .
In Figure 6D, a quadratic regression was performed on the experimental data. The non-linear regression has an R 2 = of 0.859 and has the following equation: The LC 50 for the TiO 2 NWs sample was calculated, replacing probit value of 5.0 in Equation (2), we obtained the LC 50 of 157 mg L −1 .
According to Murali et al. [9], our reported LC 50 values are within the range of the other compared assays of TiO 2 NPs in D. magna, this range was reported to be between 118 mg L −1 and 218.79 mg L −1 . Table 3 lists the LC 50 values for some TiO 2 nanosystems found in the literature. Similarly, Johari and Ashagari have reported that the LC 50 was greater than 200 mg L −1 [10]. However, only poor references considered the importance of the EHD when comparing the LC 50 . The values reported by Murali et al. [9] and Gökçe et al. [37] referenced only the mean particle size, but direct exposition occurred with suspended NPs, indicating that mean hydrodynamic size needs to be determined. In our case, we used short exposure times of 24 h because of the fast response of TiO 2 nanosystems to the adsorption of heavy metals, as, for example, in [16,39]. Hence, the 24-h LC 50 gave us insights into the concentration limits for quick water treatment and short exposure time for the D. magna individuals under the tested concentrations. More importantly, the LC 50 determination was observed to depend on the solution preparation method. When the method of stock solution preparation is the same, that is, by sonication [10], it was possible to determine LC 50 values. However, the studied particle sizes are much larger. A notable difference is found in [8], in which the LC 50 could not be found by sonication, having applied concentrations of up to 500 mg L −1 and using a nanoparticle size greater than 100 nm. However, when the solution was prepared by filtration [8], the LC 50 was found to be 5.5 mg L −1 at a very low concentration, using the average NPs size of 30 nm. Hence, solubility of TiO 2 NPs is an important parameter for the accurate determination of LC 50 values. In our case, the zeta potential values of TiO 2 nanosystems are both~−20 mV at pH = 7, indicating high colloidal stability. Figure S2 shows the typical growth in the morphological parameters that one would expect when they are not exposed to the TiO 2 sample, that is, the growth that D. magna should have naturally. Figure S3 shows the optical microscopy image of a negative control D. magna specimen on the first and the last day.

Morphological Analysis
In the boxplot presented in Figures 7 and 8, the morphological parameters are illustrated, a significant change in the heart morphological parameter was evidenced for both TiO 2 samples, this may indicate a stress level of D. magna, while the tail morphological parameter does not show substantial significance for both TiO 2 samples. Regarding the body morphological parameter, it is shown that there is not much significance for the TiO2 NWs in two of the five concentrations, so there is no effect on their growth as concluded by Lee et al. [40], while for the TiO2 NPs there is a significance considerable in four of the five concentrations in opposition to Lee et al. [40]. In the morphological parameter antenna, no noticeable significance is observed in the five tested concentrations of both TiO2 nanosystems. Therefore, the erratic behavior in the swimming of D. magna is not confirmed to be due to this malformation. This is also corroborated by the observations of Lovern et al. [8]. Figures 9A-F and 10A-F show the comparison of D. magna when they are exposed to both TiO2 samples at the tested concentrations. As can be seen, no apparent malformations were directly noticed when analyzing the optical images and no residuals of the TiO2 nanosystems were noticed on the body surface. Regarding the body morphological parameter, it is shown that there is not much significance for the TiO 2 NWs in two of the five concentrations, so there is no effect on their growth as concluded by Lee et al. [40], while for the TiO 2 NPs there is a significance considerable in four of the five concentrations in opposition to Lee et al. [40]. In the morphological parameter antenna, no noticeable significance is observed in the five tested concentrations of both TiO 2 nanosystems. Therefore, the erratic behavior in the swimming of D. magna is not confirmed to be due to this malformation. This is also corroborated by the observations of Lovern et al. [8]. Figures 9A-F and 10A-F show the comparison of D. magna when they are exposed to both TiO 2 samples at the tested concentrations. As can be seen, no apparent malformations were directly noticed when analyzing the optical images and no residuals of the TiO 2 nanosystems were noticed on the body surface.

After Exposure Properties of TiO2 Nanomorphologies
Refined X-ray diffractograms for TiO2 samples after performing ecotoxicological experiments are given in Figure S4. In Figure S4A the characteristic Bragg's peaks of TiO2 anatase are shown after the ecotoxicity experiment. These were identified using Match v3 software, confirming the crystalline phase of anatase (# 900-8214). However, some new Bragg's peaks, not related to anatase, were observed [42]. A possible explanation for these Bragg's peaks is the presence of sodium chloride or another minor impurity, though this cannot be elucidated with accuracy due to the predominance of TiO2 anatase (>90 wt. %). In Figure S4B, the refined X-ray diffractogram for TiO2 NWs (after the ecotoxicity experiment) permitted the identification of H2Ti3O7 (# 433-6946). This crystalline phase is like that shown in [43,44], wherein H2Ti3O7 is the product of the deionization process of

Reproduction Rate
The reproduction rate of D. magna occurred in the negative control in the ninth day with 61 pups born. On day 12, however, 40 pups were born, and finally on the fifteenth day, three pups were born giving 104 individuals born (control) in parallel to the exposure experiments. Regarding the individuals of D. magna exposed to the TiO 2 samples, no reproduction was observed in the period of the ecotoxicity experiment with TiO 2 NWs. In case of TiO 2 NPs, only two concentrations reported neonates, they were 300 mg L −1 (25 neonates) and 600 mg L −1 (20 neonates). It was also found from the morphological experiments that there was a delay in the reproduction of these individuals. Significant inhibition of growth and reproduction has been observed in chronic toxicity studies with TiO 2 [41], while for the D. magna exposed to TiO 2 there was a delay in their reproduction, this was observed due to the translucent properties of the crustaceans, which were monitored with the help of an optical microscope, and through observation of the eggs on the specie incubation chamber on the last day.

After Exposure Properties of TiO 2 Nanomorphologies
Refined X-ray diffractograms for TiO 2 samples after performing ecotoxicological experiments are given in Figure S4. In Figure S4A the characteristic Bragg's peaks of TiO 2 anatase are shown after the ecotoxicity experiment. These were identified using Match v3 software, confirming the crystalline phase of anatase (# 900-8214). However, some new Bragg's peaks, not related to anatase, were observed [42]. A possible explanation for these Bragg's peaks is the presence of sodium chloride or another minor impurity, though this cannot be elucidated with accuracy due to the predominance of TiO 2 anatase (>90 wt. %). In Figure S4B, the refined X-ray diffractogram for TiO 2 NWs (after the ecotoxicity experiment) permitted the identification of H 2 Ti 3 O 7 (# 433-6946). This crystalline phase is like that shown in [43,44], wherein H 2 Ti 3 O 7 is the product of the deionization process of Na 2 Ti 3 O 7 . The corresponding Bragg's angle positions for identified crystalline phases are given in Tables S3 and S4. On the other hand, the brookite phase did not appear in the phase identification using the Match v3 software, since no characteristic peak of this TiO 2 polymorph appears in the X-ray diffractogram [42]. This may be due to the aqueous solution exposure (culture solution) with D. magna that causes a segregation or dilution for this secondary phase [45]. However, this phase was corroborated by analyzing the selected area electron diffraction (SAED) pattern. Figure 11a-d shows the SAED pattern for the recovered samples with their respective indexed rotational average patterns. By analyzing before and after the structural properties, it was possible to confirm the presence of the brookite phase (as a minority phase). Hence, the fingerprints indexes confirmed the presence of anatase TiO 2 and brookite phases before and after ecotoxicological experiments. Figure 12a-p shows the EDS mapping images that was performed to corroborate the total weight composition for the samples and to confirm the presence of Ti and O elements homogenously distributed in the samples. Figure 12 depicts the EDS mapping images that were used to validate the overall weight composition of the samples and the existence of Ti and O elements that were evenly distributed in the samples. Table 4 shows the quantitative weight composition of the four samples. After ecotoxicological investigations, there was a change in the weight composition of TiO 2 NPs for Ti and O. This was mostly due to the experiment conditions and D. magna exposure (minority phases detected by X-ray diffraction). In contrast, no significant changes in TiO 2 NWs composition were observed following the biological studies. (SAED) pattern. Figure 11a-d shows the SAED pattern for the recovered samples with their resp indexed rotational average patterns. By analyzing before and after the structural p ties, it was possible to confirm the presence of the brookite phase (as a minority p Hence, the fingerprints indexes confirmed the presence of anatase TiO2 and br phases before and after ecotoxicological experiments.    Table 4 shows the quantitative weight composition of the four samples. After ecotoxicological investigations, there was a change in the weight composition of TiO2 NPs for Ti and O. This was mostly due to the experiment conditions and D. magna exposure (minority phases detected by X-ray diffraction). In contrast, no significant changes in TiO2 NWs composition were observed following the biological studies. The O-K edge and Ti L2,3 edge EELS spectra for the anatase TiO2 NPs (before and after) are given in Figure 13a. Two peaks at 455 and 461 eV are related to L3 and L2 transitions. While the O-K edge exhibited four marked peaks given by capital letters (A-D) in Figure 13b. For both TiO2 NPs, four strong peaks in the O-K edge were seen at (A) 531 eV, (B) 540 eV, (C) 545 eV, and (D) 568 eV. All of these peaks were anatase TiO2 NPs [46,47]. There were no further peaks associated with other TiO2 phases before or following TiO2 NPs. Rutile is frequently distinguished by three extra peaks positioned after the A and B peaks that were not seen in the EELS spectra [46]. For TiO2 NWs in Figure 13c,d, energy peaks located at 532 eV, (B) 540 eV, (C) 543 eV, and (D) 566 eV assigned to TiO2 polymorphs were found, indicating that, despite the different morphologies, both systems re- The O-K edge and Ti L 2,3 edge EELS spectra for the anatase TiO 2 NPs (before and after) are given in Figure 13a. Two peaks at 455 and 461 eV are related to L 3 and L 2 transitions. While the O-K edge exhibited four marked peaks given by capital letters (A-D) in Figure 13b. For both TiO 2 NPs, four strong peaks in the O-K edge were seen at (A) 531 eV, (B) 540 eV, (C) 545 eV, and (D) 568 eV. All of these peaks were anatase TiO 2 NPs [46,47]. There were no further peaks associated with other TiO 2 phases before or following TiO 2 NPs. Rutile is frequently distinguished by three extra peaks positioned after the A and B peaks that were not seen in the EELS spectra [46]. For TiO 2 NWs in Figure 13c,d, energy peaks located at 532 eV, (B) 540 eV, (C) 543 eV, and (D) 566 eV assigned to TiO 2 polymorphs were found, indicating that, despite the different morphologies, both systems retained their chemical structure (before and after ecotoxicological experiments).

Perspectives
One important subject for future research is the evaluation of how TiO2 NPs modifies the bioaccumulation of heavy metals in D. magna. Poor references are available in the literature focusing on this issue. For example, Wang et al. [48] innovatively showed that bioaccumulation of heavy metal increases to 85% in the presence of TiO2 NPs, and that this increase was correlated with the heavy metal's physical properties. Hartmann et al. [49] studied how TiO2 NPs affected the toxicity of cadmium to aquatic organisms. While the NPs could potentially carry cadmium, their addition did not change the toxicity to the studied organisms. However, more research is needed in this direction to understand the role of nanoparticle-bound versus soluble cadmium in uptake and toxicity. Finally,

Perspectives
One important subject for future research is the evaluation of how TiO 2 NPs modifies the bioaccumulation of heavy metals in D. magna. Poor references are available in the literature focusing on this issue. For example, Wang et al. [48] innovatively showed that bioaccumulation of heavy metal increases to 85% in the presence of TiO 2 NPs, and that this increase was correlated with the heavy metal's physical properties. Hartmann et al. [49] studied how TiO 2 NPs affected the toxicity of cadmium to aquatic organisms. While the NPs could potentially carry cadmium, their addition did not change the toxicity to the studied organisms. However, more research is needed in this direction to understand the role of nanoparticle-bound versus soluble cadmium in uptake and toxicity. Finally, experiments at molecular levels will also be needed to improve understanding and obtain a total ecotoxicological evaluation.

Conclusions
Two different commercial TiO 2 nanomorphologies were characterized to understand their ecotoxicological properties in D. magna. Refined X-ray diffractograms showed that the TiO 2 NPs sample has a monophasic anatase phase. Meanwhile, the TiO 2 NWs biphasic sample allowed us to identify and elucidate brookite (tetragonal) and protonic trititanate (monoclinic) phases. From the quantitative analysis, 63.5 wt. % corresponded to protonic trititanate and 36.5 wt. % for brookite. Raman spectroscopy proved the presence of both phases through their optical vibrational modes. DLS allowed the calculation of the effective hydrodynamic sizes at various pH and the zeta potential values of ca. −20 mV for both TiO 2 nanomorphologies, which are important parameters to establish the LC 50 and are not often studied when determining this value. The zeta potential was observed to increase in an alkaline medium for TiO 2 samples. This means that a negative potential can favor the adsorption of divalent cations in polluted water. However, this can represent a flaw because changing the pH can affect the D. magna habitat; thus, future evaluations in the LC 50 will be needed. Therefore, the LC 50 values must be first determined. TEM images and statistical analysis revealed no morphological changes for TiO 2 species (before and after ecotoxicological experiments). The ecotoxicological impact of TiO 2 on the environmental bioindicator was slightly higher for TiO 2 NWs than TiO 2 NPs, despite their close LC 50 values of 157 and 166 mg L −1 , the mortality in TiO 2 NWs was high and equivalent to 50% for the three of five concentrations but mortality in TiO 2 NPs was only above 50% in one of the five tested concentrations. These nanomaterials also caused a delay in D. magna reproduction, because, in favorable conditions (negative control), the neonates began to be born on day nine, having 104 D. magna individuals counted for control (after 16 days). However, those daphnids exposed to TiO 2 NWs had no neonates at the end of the experiments under the tested concentrations. On the contrary, neonates were only born in the last day of the experiments for 300 mg L −1 (25 neonates) and 600 mg L −1 (20 neonates) TiO 2 NPs. This may represent an important disadvantage that must be carefully evaluated in future works and damage comparison with studies at molecular levels will be further necessary. In terms of morphological metrics, it was found that the heart size for D. magna varies significantly when exposed to TiO 2 samples at four of the five tested concentrations. It was also observed that there was no change in D. magna swimming because there was no overall significant change in the antenna parameter after TiO 2 nanomorphologies exposure, which is the morphological feature that permits D. magna's distinctive jump. As a result of this research, we can infer that the toxic effects of TiO 2 NWs are more severe than those of TiO 2 NPs. Finally, we went beyond the ecotoxicological experiments by analyzing the recovering samples by X-ray diffraction, SAED, EDS mapping, and EELS, revealing that the structure and morphological properties of TiO 2 nanomorphologies are kept after the biological experiments. This represents a potential improvement, since they can be recycled and stored for future applications, such as water remediation, below the determined LC 50 values.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/nano13050927/s1. Figure S1: Graph of EHD vs. pH for TiO 2 NPs (anatase), each point is the result of the measurement with the highest baseline index; Figure S2: Box plot for all morphological parameters (tail, body, heart, antenna and eye) for the negative control for both the first day and the last day; Figure S3   Data Availability Statement: The original data related to this research can be asked for any time to the corresponding author's email: juan.ramos5@unmsm.edu.pe.